Mechanical stimulation releases 5-HT from enterochromaffin cells (EC) to trigger a secretomotor reflex. Our animal studies indicate that purines (ATP/ADP/ADO) operate at all levels of the reflex. Purinergic mechanisms are potential therapeutic targets in IBD, IBS or diarrheal disorders; but chronic inflammation or GI disease can alter purine gene expression to disrupt gut function. Despite implications for translational neurogastroenterology, little is known about purines in human gut reflexes or alterations in IBD or IBS. Development of effective treatments is hampered by our poor understanding of human gut neurobiology and neuroplasticity and difficulties in studying it. Our data support a novel Purinergic hypothesis that 'Purines are critical determinants of mechanosensory reflexes in human gut': EC cells express P2X3 and P2Y1R and their activation by ATP co-released with 5-HT cause amplification of 5-HT release evoked by mechanical stimulation. ATP-gated P2X3 or metabotropic P2Y1R are positive autocrine signals of 5-HT release. P2X3 activation contributes to speed and sensitivity and P2Y1 exerts slow-sustained amplification of 5-HT release. Negative autocrine signals are transduced through P2Y12 and A3R. 'Plurichemical' purinergic transmission occurs in the human ENS. 5-HT (and ATP) release triggers an ENS reflex where multiple purines act at these receptors to modulate neurogenic secretion of fluid and electrolytes. ADO inhibits immune activation of the ENS by biopsy supernatants (SUP). In IBD (or IBS) heightened immune-neural / EC activation and purinoceptor plasticity or altered purine handling mechanisms is associated with disturbed gut function. We developed novel, cutting-edge in vitro techniques for critical evaluation of purinergic mechanisms in human gut reflexes - molecular, Ca2+ imaging or electrochemical detection is used to study purinergic autocrine mechanisms of 5-HT release in BON (EC) cells, naive EC cells in intact mucosa or EC cells isolated by FACS sorting. LSM Ca2+ neuroimaging provides adequate temporal and spatial resolution to study neural circuit behavior in human ENS in normal or inflamed surgical tissue (or biopsy); this is complemented by IHC, radioligand, and molecular approaches. Secretomotor function is evaluated by studies on neurogenic secretion / ISC responses to drugs. A large volume of clinical cases and pilot data in >265 patients support Aim 1. To identify purinergic components of mechanosensory transduction in human EC cells and IBD Aim 2. To investigate plurichemical and purinergic transmission in the human ENS Aim 3. To determine in ENS whether functional and molecular interactions occur between inflammatory/immune mediators and purinergic signaling mechanisms in IBD (and IBS) Aim 4. To identify purinergic pathways in neurogenic secretomotor function and disturbances in function in IBD Studies will fill a critical gap in our knowledge of human ENS/purinergic signaling, and establish purines as critical regulators of 5-HT release, neural activity, neurosecretion and mechanosensory reflexes; immune- neural activation and plasticity in IBD/IBS - providing a sound basis for further translational studies. PUBLIC HEALTH RELEVANCE: Inflammatory Bowel Diseases (IBD) and functional disorders like Irritable bowel syndrome (IBS) are associated with significant morbidity and economic burden. Development of effective treatments is hampered by our poor understanding of human gut neurobiology, neuroplasticity, species differences in extrapolating animal data to humans and difficulties in studying it. Purines (ATP/ADP/ADO) are potential therapeutic targets in IBD, IBS or diarrheal disorders. Studies in human gut tissues or biopsy/supernatants will reveal purinergic mechanisms of function and dysfunction in IBD and IBS - Studies may reveal novel therapeutic targets.